Initial steps in the opening of a Shaker potassium channel

Abstract

The structural model of a KV (K+-selective, voltage-gated) channel in the open state is known (Protein Data Bank ID code 2R9R). Each subunit of the channel has four negatively charged residues distributed in the transmembrane segments S1, S2, and S3 that bind to and facilitate the movement within the membrane of the positively charged, voltage-sensing residues of S4. When extrapolated to the closed state, the two outermost negatively charged residues are exposed to extracellular fluid and not bound to S4 residues, all of which have theoretically been driven inward by voltage. If this closed state model is correct, these residues are available to bind external cations. We examined the effects of La3+ on voltage-gated Shaker K+ channels. Addition of the trivalent cation La3+ (50 μM) extracellularly markedly prolongs the lag that precedes channel opening and slows the subsequent rise of K+ current (IK) at all voltages. Decay kinetics of IK at negative voltages are unaltered. Gating current (Ig) recorded from a nonconducting mutant shows that La3+ reduces the initial amplitude of Ig nearly twofold. We postulate that, in the resting state, La 3+ binds to the unoccupied, outermost negative residues, hindering outward S4 motion, thus increasing the lag on activation and slowing the rise of IK. In the activated state, La3+ is displaced by outward movement of arginine residues in S4; La3+, therefore, is not present to affect channel closing. The results give strong support to the closed state model of the KV channel and a clear explanation of the effect of multivalent cations on cellular excitability.